Surprisingly, platelets are capable of many cellular processes (i.e., RNA splicing, translation, glycosylation, endocytosis); however, their effects on platelet function are often unclear. As circulating sentries, platelets sample the vasculature via endocytosis. Some of the endocytosed cargo (i.e., fibrinogen) traffics to granules; however, other material initiates platelet responses. In this proposal, we show that pathogen endocytosis induces immuno- activation of platelets. This likely contributes to the increased cardiovascular risks associated with persistent infections, (as seen in HIV-1/AIDS patients). In this application, we build on our novel findings, hypothesizing that platelet endocytosis is critical for innate immune response to viral infections. To test this hypothesis, we focus on how platelet endocytosis facilitates Toll-like Receptor (TLR)-based signaling in an animal model that mimics chronic HIV-1/AIDS. Using novel, genetically-altered mice (Arf6-/-, VAMP-3-/-, and Syntaxin-2/4-/-), which are defective at different endocytic steps, we will address the roles, routes, and mechanisms of platelet endocytosis, with specific focus on how platelets interact with HIV-1 virus. We propose two specific aims: Aim 1. Determine the effects of defective platelet endocytosis on innate immune responses in a model of viremia. Aim 2. Determine the mechanisms and routes of the platelet endocytosis system. Our proposal advances platelets as active monitors of the vasculature, continuously interacting with and interpreting their environment while circulating. In this scenario, platelets use endocytosis to continuously sample their microenvironment and, through endocytic trafficking, process and evaluate cues that initiate responses to what the platelets have endocytosed. Our work will yield insights into platelet function during systemic infections, thus reshaping the uses of anti-thrombotic therapies. Finally, understanding platelet endocytosis will uncover novel strategies to increase the efficiency of loading platelets with therapeutics that can be used to treat CVD.